This invention is in the fields of biochemistry, pharmacology, and anti-viral agents.
There is a major need for improved methods and agents to prevent the spread of acquired immunodeficiency syndrome (AIDS), which is caused by a virus known in America as the human immunodeficiency virus (HIV). Even though the last letter in "HIV" stands for "virus", it is often called the HIV virus, for convenience. It is also called the AIDS virus, and some reports (especially those published prior to 1988) call it the lymphadenopathy virus (LAV) or the HTLV-III virus, which were the names originally used by the research groups in France and the U.S. that isolated it. There are two major strains of the HIV virus, usually designated as HIV-1 and HIV-2.
An excellent introduction to the AIDS epidemic and the HIV virus, with outstanding illustrations and photographs, is provided in the October 1988 issue of Scientific American, which was entirely devoted to AIDS. Books that contain detailed information with citations to thousands of scientific and medical articles include Gottlieb et al 1989, Cohen et al 1990, Putney and Bolognesi 1990, and Levy 1989.
Briefly, the HIV virus contains an outer spherical envelope made of a lipid bilayer membrane. Directly inside the outer envelope is a second sphere comprising a protein called p17. All HIV structural proteins (p17, p24, gp41, and gp120) are named after their molecular weight in kilodaltons.
A glycosylated protein called gp41 is embedded in the lipid envelope. A second glycosylated protein called gp120 is non-covalently coupled to the gp41 protein and extends outward from the spherical envelope. Based on electron micrographs, several dozen copies of the gp120 protein appear to be coupled to each HIV particle.
The gp120 protein binds to a human protein designated as CD4 (sometimes referred to as the T4 protein, since it is encoded by a gene known as the T4 gene). The binding reaction is a non-covalent reaction of a type that is often called affinity binding; similar affinity binding reactions occur between antibodies and antigens. The CD4 protein appears on the surfaces of certain types of T-lymphocytes, and in lesser quantities on the surfaces of certain other cells, including monocytes that give rise to white blood cells known as macrophages, glial cells in the brain, and chromaffin cells in the intestine. The binding reaction between the viral gp120 protein and the cellular CD4 protein is very tight; this allows the viral particle to attach itself securely to cells that have the CD4 protein on their surfaces. The cellular entry process may also involve a human protein called CD26, which apparently interacts with a loop of the gp120 protein that becomes exposed after the gp120 protein binds to the CD4 protein.
The viral genes can enter a human cell by any of several processes (Weber and Weiss 1988). In one process, the lipid membrane of the virus fuses with the cell membrane, which is also a lipid bilayer. In an alternate process, an attached virus might be taken into a cell by a process called receptor-mediated endocytosis, in which the viral particle is transported into the cell in a lipid sac that is digested after it enters the cell. In another reported mechanism, in which cells that do not have CD4 receptors can be infected, HIV-infected lymphocytes bind to epithelial cells and inject HIV particles directly into the epithelial cells (Phillips and Bourinbaiar 1992; Pearce-Pratt and Phillips 1993; Zacharopoulos et al 1992). These tests utilized epithelial cells from the intestine, presumably to model the transmission of HIV after anal intercourse, which deposits infected lymphocytes in the colon. The viral assays in those reports were performed seven days after the infection reaction; however, by the time an epithelial cell reaches the surface of a mucous membrane inside the vagina of a woman of reproductive age, the cell remains there only about 96 hours (4 days) before being sloughed off (Averette et al 1970; Ferenczy and Guralnick 1979). That time period is even lower if the woman is taking estrogen. In addition, even though epithelial cells retain their nuclei as they reach a mucous membranes surface (in contrast to epidermal cells, which lose their nuclei before reaching the surface), epithelial nuclei become "pyknotic" (i.e., they become compacted and inactive, in a manner resembling the nuclei of dead or dying cells) as the cells reach an epithelial surface. For both of these reasons, most epithelial cells that have reached a surface layer inside a vagina probably cannot be used by HIV to establish an active reservoir of cells that can support viral replication. It should also be noted that none of the reports co-authored by Phillips and his coworkers evaluated the effects of zinc on the cell--cell binding processes they studied, even though zinc is present in high concentrations in semen and significantly alters the behavior of cell membranes.
The core of an HIV particle contains a protein shell with a conical shape, made of p24 protein units. Unlike most viruses that are enclosed within protein capsids, the p24 protein shell usually is not referred to as a capsid, apparently because the p24 shell is not an enclosed structure (it is open at one end) or because it is surrounded by an outer envelope.
The p24 shell contains two identical strands of single-stranded RNA roughly 9.7 kilobases (kb) long each, and several viral enzymes. One such enzyme carried in the core is reverse transcriptase (RT). When released from the viral core after the virus has entered a cell's cytoplasm or nucleus, the DNA polymerase domain of the RT enzyme uses the viral RNA as a template to synthesize a first strand of DNA. In the next step, an RNase domain of the RT enzyme (which may be an independent polypeptide) digests the viral RNA strand to get it out of the way. In the third step, the viral DNA polymerase synthesizes a second strand of DNA, using the first DNA strand as a template. These three steps generate double-stranded DNA (dsDNA) carrying the viral sequence.
After dsDNA synthesis, a second viral enzyme called integrase (which also is apparently carried inside the viral capsid) helps the viral dsDNA become integrated (i.e., inserted by means of stable covalent bonding) into a chromosome of the cell. The viral dsDNA, when inserted into the chromosome, is called a provirus; some articles apparently also refer to viral dsDNA in free, non-integrated form as a provirus as well.
Inside the nucleus, the proviral dsDNA can remain in an inactive (dormant or latent) state for very long periods. If and when the virus becomes active, the viral genes are transcribed into RNA, primarily by normal cellular enzymes with additional involvement by several viral regulatory genes including tat, rev, and nef.
Some of the viral RNA functions as messenger RNA (mRNA); it is translated into polypeptides by the cell's ribosomes. A viral protease enzyme, which apparently is carried inside the viral core, cleaves the viral polypeptides in a specific manner, releasing functional viral proteins from the longer precursor polypeptides. The viral proteins, along with viral RNA and lipids, are assembled into new viral particles which are released by the cell. Some types of cells release newly generated HIV viruses when the cells lyse (i.e., when the cells are broken apart); other cell lines release HIV by means of a budding process that does not kill the cells.
Zinc Studies by Prior Researchers
A great deal of study and countless proposals have been devoted to potential ways to treat HIV infection, and every known step in the process of infection or replication has been proposed as a target for intervention by drugs that might be able to stop or slow down the virus. Some of those prior efforts and proposals briefly focused on various zinc compounds; however, none of the zinc studies in the prior art encountered any success in the assays used by those researchers, so those researchers lost interest in zinc compounds and turned their attention to other candidates.
For example, when the Applicant submitted samples of several zinc salts (including zinc acetate and zinc gluconate) to the National Cancer Institute for evaluation in a standardized screening test against the HIV virus, the scientists in charge of the NCI's screening program returned the sample without even opening it. The NCI's letter of response, dated Aug. 8, 1991, stated as follows: "After careful consideration, we have decided not to test your zinc salts in our AIDS-antiviral assay . . . We have tested 36 zinc-containing compounds in our in vitro AIDS screen, including zinc gluconate (test results of this inactive compound, NSC 619899, are enclosed . . . ). None of these materials has demonstrated any activity worth pursuing . . . Based on these results, we don't believe that your compounds will show activity in our assay." This is an authoritative statement that, based on their chosen assays, researchers skilled in the art of HIV research did not believe that zinc salts could serve as effective agents against HIV infection.
After analyzing the details of the assay protocols used by other researchers, and after studying the toxicological aspects of zinc when unnaturally high concentrations contact lymphocytes, the Applicant recognized why assays carried out by other researchers did not reveal any substantial beneficial effects of zinc. Briefly, the assays used by the National Cancer Institute evaluated zinc as an agent in the bloodstream; they did not make any effort to evaluate zinc as a topical agent on genital surfaces during intercourse. The assays, described in Weislow et al 1989, involve combining lymphocyte cells, HIV particles, and a candidate antiviral agent simultaneously, then determining whether the antiviral agent protected the lymphocytes.
Such assays may be effective in studying agents that enter the blood and act as viricides or site-specific receptor blockers, but such assays could not identify the antiviral activity of topical agents that do not penetrate the skin or enter the bloodstream. This is especially true in the case of zinc. Zinc concentration in the blood is tightly regulated by a number of proteins and cellular mechanisms (as discussed below), and higher concentrations which do not naturally occur in the blood are relatively toxic to such lymphocytes. Therefore, assays such as the ones described in Weislow et al 1989 or Resnick et al 1990 did not (and could not) adequately evaluate or identify the antiviral properties of zinc applied topically in a lubricant rather than used as an injected or oral drug.
To overcome those problems, the Applicant developed a two-stage assay that attempts to model what would occur inside the vaginal cavity, but outside the skin, after sexual intercourse between an HIV-positive person and an uninfected partner. These assays use zinc concentrations that can be tolerated on the genital surfaces without irritation or toxicity, even though such concentrations would be toxic to lymphocytes if injected into the blood. During the initial incubation step, which is relatively brief, the zinc is given an opportunity to react with viral particles, with no lymphocytes present. Subsequently, the zinc-virus mixture is diluted to reduce the zinc concentration to levels that are not toxic to lymphocytes, and lymphocytes are then added and incubated with the zinc and virus for 20 days or more. During this second stage of incubation, zinc-treated viruses were shown to be non-infective, while viruses diluted in an identical manner remained highly infective. Those two-stage incubation tests are discussed in more detail below, under the description of the invention.
The following sections provide additional data on zinc toxicology, and on zinc's ability to stabilize membranes and promote the healing of skin deficits.
Zinc Physiology and Toxicology
Zinc is an essential mineral, found in every form of life on earth. Indeed, because of its role as an essential component of certain proteins that interact with DNA, it is believed to be present in every single cell on earth.
In mammals, the great majority of zinc in the body is in skeletal muscle and bone. Blood zinc constitutes less than 0.5% of total zinc in the body, and that fraction is mostly contained inside blood cells or is bound to cell surfaces. In blood plasma, which contains about 1 microgram (ug) of zinc per gram of blood, zinc is bound to various proteins, including albumin, alpha macroglobulin, and transferrin (Vallee 1988; Cousins 1989). These binding reactions are reversible, and they establish an equilibrium between ionic and protein-bound zinc. High zinc levels also stimulate the expression of metallothionein, which tightly chelates zinc (Sadhu and Gedamu 1990). In addition, secretions from the kidneys and pancreas, which are stimulated if zinc levels begin to rise, cause the excess zinc to be excreted in the urine and feces. All of these factors allow the concentration of zinc in blood to be tightly regulated and constrained within a narrow range even though zinc intake can vary widely.
Due to these factors, the toxicity of zinc inside the body is very low. Toxicity problems can arise in unusual situations; for example, inhalation of zinc fumes by metalworkers can lead to a condition called metal fume fever, and genetic defects render certain people unable to metabolize zinc properly. However, such problems are rare, and in healthy people, toxicity caused by excessive zinc is virtually nonexistent and most of the scientific and medical literature on zinc toxicity actually relates to zinc deficiencies, rather than excess zinc.
As stated in Vallee and Falchuk 1993, an extensive review article, "unlike other metals, including those of the IIB series, zinc is virtually nontoxic. The homeostatic mechanisms that regulate its entry into, distribution in, and excretion from cells and tissues are so efficient that no disorders are known to be associated with its excessive accumulation, in contrast to iron, copper, mercury, and other metals. Second, its physical and chemical properties, including its generally stable association with macromolecules and its coordination flexibility, make it highly adaptable to meeting the needs of proteins and enzymes that carry out diverse biological functions. These and yet other chemical properties form the basis for the extensive participation of zinc in protein, nucleic acid, carbohydrate, and lipid metabolism, as well as in the control of gene transcription and other fundamental biological processes."
Other review articles reach similar conclusions; for example, Leonard et al 1986 concludes that "Toxicity of zinc is low . . . zinc is not mutagenic and has little, if any clastogenic properties . . . zinc is not teratogenic; it can, in fact, avert teratogenicity of other agents. Conversely, zinc deficiency may be harmful." Other toxicologic and physiologic reports include Mills 1989, Calesnick and Dinan 1988, Fosmire 1990, and Bach 1981.
In healthy men, zinc is present in semen at concentrations of 100 to 500 ug/g, and in prostate fluid at concentrations up to 1000 ug/g (Eliasson and Lindholmer 1971; Fair et al 1976; Homonnai et al 1978; Marmar et al 1980). These levels are extraordinarily high compared to blood concentrations of only about 1 ug/ml. In prostate fluid, zinc exerts an antimicrobial effects, to combat infection of a fluid that cannot be directly protected by the immune system (Fair et al 1976). In undiluted semen, zinc suppresses the respiratory activity and motility of sperm cells (Eliasson 1971; Paz et al 1977). Apparently, this allows the sperm cells to stay in a quiescent state, storing and conserving their energy until it is needed. After ejaculation, the zinc is diluted by the female's vaginal fluids and it binds to proteins and other cells inside the vagina. This decreases the concentration of sperm-bound zinc; this, in turn, allows the respiratory activity and motility of the sperm to increase.
Some researchers refer to zinc as a "heavy metal" (e.g., Hedberg et al 1991). This nomenclature uses an arbitrary classification that refers to any element heavier than iron (molecular weight of 56) as a "heavy metal." However, since the phrase "heavy metal" implies "toxic and dangerous" to many readers, zinc (with a molecular weight of 65, close to that of iron) should be regarded as a transition metal or an essential mineral, comparable to iron or manganese.
Membrane Stabilization and Skin Healing Properties
Zinc is widely used as a soothing and healing agent in numerous types of ointments, creams, powders, and other formulations that are applied topically (i.e., spread upon a skin surface). When applied to skin incisions in scientifically controlled studies, it promoted epidermal cell growth and healing (Agren 1990). Zinc is the main active ingredient in ointments used to treat diaper rash (such as Desitin.TM. baby ointment, sold by Pfizer) and decubitis ulcers (bedsores), in calamine lotion, and in numerous sunblocking creams, antiperspirants, and antifungal agents. Parents have been spreading zinc onto the genitals of their babies for decades, to cure diaper rash; this is strong evidence of its complete absence of toxicity, and of its soothing and healing properties even when applied to highly sensitive areas and to areas that are irritated, inflamed, and in need of soothing.
Some topical formulations contain more than 30% elemental zinc by weight. Most topical formulations use zinc oxide, which gradually solubilizes and releases free zinc ions when it contacts body fluids (Agren 1990).
On a molecular and cellular level, zinc stabilizes and protects cell membranes by mechanisms such as protecting sulfhydryl groups against oxidation and inhibiting the formation of free radicals that randomly attack and degrade membrane-forming lipids and other biomolecules (Chvapil 1973 and 1976; Mahadevan et al 1990; Bray and Bettger 1990; Pasternak et al 1992; Kaszuba and Hunt 1990). Zinc can also suppress the leakage of metabolites out of cellular pores or lesions created by various bacteria and viruses. In addition, zinc also increases the activity of various enzymes that help cells withstand stress, such as glucose transporters (Pasternak 1990), ecto-nucleotidases (Meftah et al 1991), and certain protein kinases (Zalewski 1991). Zinc also increases the integrity of internal multicellular membranes, such as blood vessel walls (Hennig et al 1992), and it appears to have a bifunctional interaction with actin; at low-to-moderate concentrations of zinc, actin increases cell membrane permeability, while at high concentrations of zinc, actin reduces membrane permeability (St. Onge and Gicquaud, 1990).
In short-term tests, zinc has been shown to be harmless or beneficial inside the vagina (Chvapil et al 1978a and 1978b; also see Williams 1980 and Chvapil 1980). These reports describe research using guinea pigs, rabbits, and human volunteers to study whether zinc would be an effective contraceptive. It was only about 80% effective on a single-event basis, so interest in its use as a potential contraceptive died out. However, during those tests, it was shown that (1) most of the zinc introduced into the vagina became bound to vaginal fluids or cells, and was washed out of the vagina within a few days by the natural flow of fluid and the exfoliation of epithelial cells from internal vaginal surfaces; (2) zinc content in vaginal tissue in treated animals was not significantly different than in control animals; and, (3) zinc did not cause any significant swelling, redness, tenderness, or histological changes to vaginal membranes.
Indeed, when introduced into the vagina along with other contraceptive agents or collagen sponges, zinc played a beneficial role; it prevented the generation of offensive smells ("malodors") that were occasionally encountered when untreated sponges were removed, and it reduced or prevented the irritation or swelling caused by such other agents in the absence of zinc. These are consistent with zinc's utility as a a mild broad-spectrum antibacterial and antifungal agent, and with its additional utility as a topical healing agent.
These beneficial effects become especially interesting in light of a report which stated that nonoxynol, a surfactant that attacks lipid membranes, actually increased the risk of HIV infection among prostitutes (Kreiss et al 1992). There is no question that nonoxynol can destroy HIV particles, by attacking the lipid envelopes which surround them. However, if used with high frequency (as occurred among the prostitutes in Kenya who were studied), nonoxynol can also create vaginal lesions, which are essentially open sores. These open sores can become entry ports for HIV particles to pass through the protective barrier of the skin and reach the bloodstream. Accordingly, among people who use nonoxynol as a contraceptive or condom lubricant, nonoxynol can be alternated with a lubricant containing zinc, to promote healing of any nonoxynol-induced lesions.
Zinc can also promote and accelerate the healing of lesions caused by other sexually transmitted diseases, including herpes and syphilis. Such other diseases have been shown statistically to increase the risk of HIV infection (e.g., Holmberg et al 1988) and it has been estimated that lesions from sexually transmitted diseases such as herpes and syphilis can increase the risk of HIV particles establishing an infection, after exposure during and after intercourse, by up to a hundred-fold. Accordingly, the ability of zinc to promote and accelerate the healing of such genital lesions is an important and highly beneficial factor in the use of zinc in a genital lubricant formulation.
Anti-Viral Activity of Zinc
Based on cell culture tests, zinc has been reported to be effective against numerous different types of mammalian viruses, including sindbis virus (Bracha et al 1976), foot and mouth disease virus (Firpo and Palma 1979), vaccinia virus (Zaslavsky et al 1979), aphthovirus (Sharma et al 1985), and rhinoviruses (Korant et al 1976A and 1976B; Godfrey et al 1988). These items are discussed in more detail in above-cited application Ser. No. 737,169, the contents of which are hereby incorporated by reference. That patent application focuses on the herpes simplex virus type 2 (HSV-2), which causes genital herpes. The use of zinc salts (primarily zinc sulfate, which causes substantial burning and irritation in most patients) to treat established herpes infections is discussed in Gordon et al 1975, Fahim et al 1980a and 1980b, Tennican et al 1979 and 1980, Wahba et al 1980, Brody et al 1981, Eby and Halcomb 1985, and U.S. Pat. Nos. 4,465,666 and 4,762,715 (Lukas et al). U.S. Pat. No. 4,407,818 (Lionelle and Staffa, 1983) also discloses a zinc oxyacetate complex which was reported to be effective against herpes. None of those references relate to AIDS or the HIV virus.
In a patent application filed under the Patent Cooperation Treaty, number WO 8702246, William Sergio suggested that the risk of infection with AIDS might be reduced by topical administration of zinc salts and other compounds that generate anions having charges greater than one. Sergio's reference to anions was mistaken, since an anion is a negatively charged ion, while zinc ions are cations (positively charged). Sergio also states that his preferred salts are zinc phosphonoformate and/or zinc tungstate; however, it is likely that the use of either of those compounds in a sexual lubricant would cause irritation and toxicity. His suggestion concerning zinc tungstate appears to be based on the tungsten compound HPA-23, which, as Sergio conceded elsewhere, has "serious side effects" (Sergio 1988). The Sergio proposal apparently never issued in any allowed patents, and it was also contradicted by data gathered by researchers at the National Cancer Institute and elsewhere, indicating that in their assays, zinc had no beneficial effect against HIV.
One object of this invention is to provide an agent and a method for reducing the risk that a person who has previously not been infected by HIV will become infected if that person has sexual intercourse with someone who carries the virus.
Another object of this invention is to disclose a method for utilizing a non-toxic, non-irritating anti-HIV agent in a carrier formulation comprising a non-irritating sexual lubricant that can be spread on the genitals during sexual intercourse to reduce the risk that an uninfected person will become infected by HIV.
Another object of this invention is to provide a sexual lubricant which contains an effective topically-active anti-HIV agent which is non-toxic and non-irritating to the genitals and urethral and vaginal membranes.
The discussion and claims which follow focus primarily on zinc salts as anti-viral agents. However, co-pending U.S. application Ser. No. 07/816,278, cited above, also contained an additional item which is incorporated herein by reference. It disclosed the use of relatively small fragments of the human CD4 peptide, which can bind to the viral gp120 protein, in a topical lubricant. One of the key aspects of that disclosure was the disclosure that such CD4 fragments could be coupled to polymeric backbones, to increase their effectiveness. This would render the CD4 fragments analogous to fishing hooks tied to a fishing line; it would only require a single CD4-gp120 binding reaction to ensnare a viral particle and reduce its motility and its ability to contact and infect susceptible cells. By contrast, prior proposals to use soluble CD4 fragments suffer from a major limitation: each soluble CD4 fragment can bind to and inactivate only a single gp120 peptide, and every HIV particle has dozens of gp120 peptides on its surfaces. As noted above, the disclosure that CD4 fragments should be attached to polymeric backbones to increase their effectiveness in a topical lubricant is incorporated herein by reference.